To understand pathogenesis, one must study the pathogen of interest within an environment that induces the expression of genes necessary for survival or for causing disease, such as the natural host or an animal model. Gene expression within a host can provide clues to the pathogenesis of an organism, including virulence factors. Although host immune response to a pathogen and the pathogen itself are often studied separately, the response of the pathogen to host immune mechanisms is often neglected. Bacteria regulate gene expression in response to environmental stimuli. Thus, it is likely that a pathogen will modulate its gene expression to survive the host immune response. In the case of Mycobacterium tuberculosis, little is known about virulence and survival mechanisms, or the impact of host factors on pathogen gene expression. As a first step in the identification of genes encoding potential virulence and survival factors it is proposed to address the question of mycobacterial gene expression in murine models with a focus on the effect of the host immune response on gene expression by this pathogen. Although there are many immune responses elicited by mycobacterial infection, the focus of this proposal will be on macrophage activation. Improved RNA subtractive hybridization methodology is proposed to study gene expression induced by macrophage activation both in vivo and in vitro. M. tuberculosis gene expression in activated and resting tissue culture macrophages will be compared. Using this system it is hoped that a population of genes induced in response to macrophage activation in vitro will be isolated. It is also proposed to assess the effects of macrophage activation on mycobacterial gene expression in vivo, using two mouse models which are deficient or delayed in macrophage activation due to defects in cytokine action. Gene expression in bacilli from wild type and gene-disrupted mice will be compared, to isolate a population of genes that are induced by macrophage activation in vivo. A positive selection hybridization procedure is proposed to identify a subset of genes commonly expressed in the in vitro and in vivo models. It is hoped that characterization of the genes commonly expressed in these model systems will provide clues for the mechanisms macrophage survival of M. tuberculosis and possibly lead to information regarding the ability of this pathogen to survive the immune response.